DNA topoisomerases are proteins responsible for controlling and maintaining the topological state of DNA the cell. They have been found in all cell types of both eukaryotes and prokaryotes and additionally in some viruses. They are involved in DNA replication, transcription, and genetic recombination. Their role in DNA metabolism has made them an important target of novel chemotherapeutic and antibiotic agents. All topoisomerases work by forming a transient break in DNA through a phosphotyrosine bond. Type I topoisomerases break one DNA strand at a time and then pass another strand through the transient break; type II enzymes break both strands and pass both strands through the break. An understanding of the detailed mechanism of action of DNA topoisomerases is now emerging. The structure of a 67 kDa amino terminal fragment of E. coli DNA topoisomerase I has allowed us to suggest a detailed mechanism of action consistent with the existing biochemical, mechanistic, and structural data. We are now in the process of testing this mechanism by studying the interactions of the fragment with short oligonucleotides and also by studying the structure of a 30 kDa fragment that contains the active site tyrosine. The study of this fragment is particularly important for our understanding of the mechanism of action as it plays a crucial role in the reaction. In collaboration with R. DiGate, we are also studying E. coli DNA topoisomerase III, a potent catenating decatenating enzyme. We have succeeded in crystallizing the intact protein although the crystals are very small and radiation sensitive. We collected data on beamline A-1 using rntation/oscillation camera and either the ADSC CCD detector from crystals of the 67 kDa and 30 kDa fragments of E. coli DNA topoisomerase I and from the intact E. coli DNA topoisomerase III. In all cases the crystals were kept at cryogenic temperatures to prolong their lifetime in the beam. We collected data on crystals of the 67 kDa amino terminal fragment of E. coli DNA topoisomerase I soaked with different oligonucleotides. We are currently refining the structure of a 5'TMP soaked crystal and also of a double soak of 3' and 5' TMP. This experiments are important to elucidate the single strand DNA bindins sites and the polarity of the DNA chain during the topological manipulations.. We also collected data on the intact E. coli DNA topoisomerase III to 2.5 _ resolution. We collected data from several crystals as the crystals decay quickly in the x-ray beam. We can only measure 2.5 _ data at CHESS; we have never seen difraction beyond 3.2 _ at any other radiation source. Data collection from crystals of topoisomerase III has presented a major technical challenge due to the sizeof the crystals (less than 100 mm thick), the relatively large unit cell (236 _ x 236 _ x 108 _, hexagonal), and the problems with radiation damage (we can only collect around 2.5-5 degrees per crystal in a random orientation).